The overall goal of this proposal is to identify and characterize cellular proteins that bind to viral hemorrhagic fever viruses. Specifically, we focus on RNA viruses from four families: the filoviruses Ebolavirus and Marburg virus; the Arenaviruses Junin virus and Lassa Fever virus; and the Bunyavirus Crimean-Congo hemorrhagic fever virus. These emerging viruses pose significant risks to human health and national security. While each differs in the types of proteins encoded, they share similar disease outcomes, causing severe, often fatal infections in humans; all are classified as NIAID Category A Priority Pathogens. Due to the high level containment needed to work with many of them, there is a significant gap in our understanding of these viruses and no specific, approved therapies are currently available. Like all viruses, these emerging viruses rely on host cell proteins in order to replicate. This dependence represents a potential Achilles heel in the virus life cycle that may be exploited to develop new approaches to treat viral infections. However, only a small number of virus-host cell protein interactions have been reported for the viruses in this study, and no systematic analysis of emerging virus-host cell protein interactions has been performed. In this project we develop and evaluate high quality maps of emerging virus-human protein interactions. In Aim 1 we use complementary yeast two-hybrid screening and co-affinity purification plus mass spectrometry approaches to identify cellular proteins that bind to emerging viruses and closely related non-pathogenic viruses from the same family. In aim 2 we identify shared and virus-specific features of the host cell interaction networks of emerging viruses. We use orthologonal protein-protein interaction assays to identify shared and virus specific interactions within each virus family. To gain confidence in these results, each assay is benchmarked against the human positive and random reference sets. We then employ informatic approaches to discover pathways that targeted preferentially by emerging viruses, develop tissue-specific virus-host cell protein interaction networks, and identify small molecule agonists or antagonists predicted to inhibit virus replication. In aim 3, we test the predictions generated through the bioinformatic analyses. A subset of high-interest cellular proteins targeted by multiple viruses will be experimentally interrogated for their contribution to infectio using RNA interference. State-of-the-art molecular techniques are then employed to determine the mechanisms by which they contribute to virus replication. Virus- and tissue-specific interactions are evaluated for their contribution to virus replication and their effect on cellular signaling pathways. Finally, small molecule inhibitors identified in our network models are evaluated for their effect on virus replication in cell culture. The data generated from this projet will improve our understanding of the functions of individual viral proteins, provide insight into the overall strategies used by viral hemorrhagic fever virus to interface with their host cells, an may lead to the discovery of new targets for treatments of emerging viruses.